Smart fluorescent proteins: Innovation for barrier‐free superresolution imaging in living cells

Abstract

During the past decade, several novel fluorescence microscopy techniques have emerged that achieve incredible spatial and temporal resolution beyond the diffraction limit. These microscopy techniques depend on altered optical setups, unique fluorescent probes, or post-imaging analysis. Many of these techniques also depend strictly on the use of unique fluorescent proteins (FPs) with special photoswitching properties. These photoswitchable FPs are capable of switching between two states in response to light. All localization precision and patterned illumination techniques-such as photo-activation localization microscopy, stochastic optical reconstruction microscopy, reversible saturable optically linear transitions, and saturated structured illumination microscopy-take advantage of these inherent switching properties to achieve superior spatial resolution. This review provides extensive analysis of the positive and negative aspects of photoswitchable FPs, highlighting their application in diffraction-unlimited imaging and suggesting the most suitable fluorescent proteins for superresolution imaging.

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

“…In the years since the discovery, significant advances have been taken in discovering or developing new fluorescent protein photoswitches and mutants with characteristics tailored for specific experiments, including super-resolution fluorescence imaging applications. Several recent reviews summarize many of these developments in the context of super-resolution imaging [41][42][43][44][45].…”

Optical Nanoscopy Tools for Biologists:Advancements of Fluorophores and Optics for High Resolution and Live Imaging

“…The switching on/off of the fluorophore molecules in random or stochastic methods should be controlled so that a small subset of labelled fluorophores switches-on in each frame. This process can be controlled either by repeatedly switching the fluorophore on and off or by switching the fluorophore on and then bleaching it [53][54][55][56] . Using this approach, the achievable resolution in stochastically switching methods is down to 10-20 nm, far better than the diffraction limit of light (~200 nm).…”

“…This improvement caused much excitement and made the single moleculebased nanoscopy methods popular among researchers that eventually led to the 2014 Nobel Prize in Chemistry. Many types of photoswitchable fluorophores are used in this method, these are reversibly or irreversibly switchable from one fluorescent state to another 2,56,57 . Both photochromic chemical dyes and genetically encoded fluorescent proteins (FPs) can be used, depending on the purpose.…”

A perspective on optical developments in microfluidic platforms for Caenorhabditis elegans research

“…PALM and STORM also require the ability to introduce photoswitchable dyes in specific target cells or sub-cellular units, which may be challenging in C. elegans. 65 Future research is likely to focus on the development of techniques that are feasible to implement in regular biology labs without special equipment and materials, and techniques that are scalable.…”